Methods, devices, and reagents for monitoring paclitaxel concentration in plasma for pharmacokinetic-guided dosing of paclitaxel

ABSTRACT

Methods, devices, and compositions for assaying therapeutic agents. In one aspect, methods, devices, and compositions for assaying paclitaxel to provide therapeutic drug monitoring guided therapy of paclitaxel.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/742,513, filed Jun. 17, 2015, which is a continuation ofInternational Application No. PCT/US2015/024578, filed Apr. 6, 2015,which claims the benefit of U.S. Patent Application No. 61/975,386,filed Apr. 4, 2014, and U.S. Patent Application No. 62/051,757, filedSep. 17, 2014, each expressly incorporated herein by reference in itsentirety.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing is 64114_SEQ_Final_2017-07-20.txt. The text file is10.5 KB; was created on Jul. 20, 2017; and is being submitted viaEFS-Web with the filing of the specification.

BACKGROUND OF THE INVENTION

Paclitaxel, originally isolated from the bark of Pacific Yew tree, hasbeen established as one of the most effective chemotherapeutic drugs fora range of cancer types including lung, ovarian, and breast cancers. Amajor limitation of paclitaxel is its low solubility and the need to beformulated in toxic organic solvents, typically polyoxyethylated castoroil and dehydrated ethanol mixtures (known as TAXOL®). To prevent thesolvent toxicity paclitaxel has been formulated with a variety ofexcipients as well as using nanoparticle delivery systems that canimprove the solubility of hydrophobic drugs such as paclitaxel.

ABRAXANE®, a paclitaxel albumin bound nanoparticle formulation wasapproved by FDA in 2005 and is currently one of the best formulations ofpaclitaxel for chemotherapy. Other systems have been investigated forthe delivery of paclitaxel or are in development, e.g., using polymericnanoparticles, lipid-based nanoparticle formulations, polymerconjugates, inorganic nanoparticles, carbon nanotubes, nanocrystals, orcyclodextrin nanoparticles (see, for example, Ping Ma et al., 2013, J.Nanomed. Nanotechnology: 4:2).

Although ABRAXANE® is a widely used chemotherapeutic agent andpractically applicable to all cancer types, the response to ABRAXANE®,however, can be as low as 20%. The relative insensitivity to paclitaxelfound in some patients could be a contributing factor to low responserate. However, this insensitivity may not the primary reason for the lowresponse rate. There is up to 10-fold variations in blood concentrationof paclitaxel monitored in clinical patients' samples when dosed at thevarious approved doses (260 mg/m² for metastatic breast cancer, 125mg/m² for pancreatic cancer, and 100 mg/m² for lung cancer (Nyman D W etal., 2005, J Clin. Oncol. 23, 7785-93). This variation suggests that thevast majority of patients are potentially dosed incorrectly with eithertoo great a concentration of paclitaxel administration, and had to betaken off the treatment, or too low a dosage administered and providingno benefit from the treatment. Even if patients are sensitive topaclitaxel, having an insufficient drug level would render themnonresponsive and the treatment ineffective. The under-dosed group isthe most vulnerable patient population, as it is difficult to determinewhether they are insensitive to paclitaxel or not administeredsufficient paclitaxel. Full pharmacokinetic (PK) profiling is the onlyapproach in such cases to provide guidance for proper drug dose based onthe individual pharmacokinetic variation.

Currently there are no available methods to perform a full PKquantitation of paclitaxel without having the patient enrolled incomprehensive clinical testing, which requires a hospital stay. Typicalduration of such PK testing may be over a 48 hour period and includesrepetitive blood drawing. Presently, the use of complex laboratoryequipment is required to analyze blood concentration of paclitaxel,including liquid chromatography/mass spectrometry (LC/MS) methods. Thesemethods are extremely costly, currently over $120/sample and theequipment cost is in the range exceeding $150K-$200K per instrument. Ithas also been demonstrated that a minimum of four data points collectedover a period of 48 to 72 hours is needed to adequately characterize thePK parameters for each particular patient. Keeping the patients inhospital for PK testing can easily push the cost to roughly $10,000 perpatient. A sufficiently powered Phase III clinical trial to demonstrateclinical efficacy for PK guided dosing would require 500 patients (250patients for BSA dosing and 250 patients for PK guided dosing). Thebioanalytical cost alone would be $1.5M (500 points×6 cycles ofchemotherapy×4 blood samplings for PK analysis×$120/sample analysis).The other components of trial would cost roughly $100,000 per patient,totaling $50M. This represents a significant barrier to obtainingmeaningful clinical data necessary to guide dose adjustment for optimumtumor response and regulatory approval of the device. The high cost ofthe analysis and instrumentation, therefore, has prohibitiveconsequences on establishing therapeutic drug monitoring (TDM) for manydrugs that have a relatively narrow therapeutic range.

Accordingly, a need remains for a simple, effective, and inexpensiveapproach to monitor the pharmacokinetics of paclitaxel in a patient,thereby appropriately personalizing the therapy to the individualpatient by informing any adjustment of the dosing strategy. The presentinvention seeks to fulfill this need and provides further relatedadvantages.

SUMMARY OF THE INVENTION

The present invention provides methods, devices, and compositions forassaying therapeutic agents. In one aspect, methods, devices, andcompositions for assaying paclitaxel are provided.

In one aspect, the invention provides a method for assaying paclitaxelin a liquid sample. In one embodiment, the methods comprises:

(a) applying a liquid sample comprising paclitaxel to a lateral flowassay device, the device comprising

(i) a sample receiving zone for receiving the liquid sample;

(ii) a detection reagent zone in liquid communication with the samplereceiving zone and downstream in flow direction from the samplereceiving zone,

wherein the detection reagent zone comprises a detection reagentdeposited thereon,

wherein the detection reagent is a paclitaxel antibody, or fragment orderivative thereof that binds paclitaxel, labeled with a detectablereporting group, and

wherein the paclitaxel antibody, or fragment or derivative thereof, hasa K_(on) from about 10⁴ to about 10⁷, and a K_(off) from about 10⁻³ toabout 10⁻⁷; and

(iii) a capture zone in liquid communication with the detection reagentzone and downstream in flow direction from the detection reagent zone,

wherein the capture zone comprises first and second capture reagentsimmobilized thereon,

wherein the first capture reagent is a paclitaxel material capable ofbinding the detection reagent (test line),

wherein the second capture reagent is an antibody capable of binding thedetection reagent (control line),

wherein the first capture reagent is positioned at a first distancedownstream in flow direction from the upstream end of the capture zone,

wherein the second capture reagent is positioned at a second distancedownstream in flow direction from the upstream end of the capture zone,

wherein the second distance is greater than the first distance, and

wherein the ratio of the first distance to the second distance is fromabout 0.0 to about 0.4, when the K_(on) is greater than about 2.0×10⁵and the K_(off) is less than about 1.0×10⁻³, and

wherein the ratio of the first distance to the second distance is fromabout 0.2 to about 1.0, when the K_(on) is greater than about 2.0×10⁴and the K_(off) is less than about 2.0×10⁻⁴; and

(b) allowing the sample to flow from the sample receiving zone throughthe detection reagent zone to provide detection reagent with paclitaxel;

(c) allowing the detection reagent with paclitaxel to flow through thecapture zone,

whereby the first capture reagent (test line) competes with the analyte(paclitaxel) for binding with the detection reagent, and

whereby the second capture reagent (control line) binds excess detectionreagent; and

(d) observing the amount of detection reagent bound to the first capturereagent (test line) relative to the second capture reagent (controlline).

In certain embodiments, the method further comprising determining thequantity of paclitaxel in the sample by quantitating the amount ofdetection reagent bound to the first capture reagent. Quantitating theamount of detection reagent bound to the first capture reagent caninclude optical density measurement.

In one embodiment, the paclitaxel antibody is 3C6. In anotherembodiment, the paclitaxel antibody is 8A10. In certain embodiments, twopaclitaxel antibodies, or fragments or derivatives thereof that bindpaclitaxel are used (e.g., 3C6 and 8A10).

In the method, the paclitaxel material is a paclitaxel antigen thatcompetes with paclitaxel for binding to the detection reagent. In oneembodiment, the paclitaxel material is a paclitaxel protein conjugate.

In the methods of the invention, distance between the sample receivingzone and the first capture reagent can be varied to optimize paclitaxeldetection sensitivity. In certain embodiments, the distance between thesample receiving zone and the first capture reagent is minimized tooptimize paclitaxel detection sensitivity.

In certain embodiments, the method further comprises observing theamount of excess detection reagent bound to the second capture reagent(control line). In these embodiments, the method may further comprisedetermining the quantity of paclitaxel in the sample by quantitating theamount of detection reagent to the second capture reagent.

To enhance sensitivity and dynamic range, the method may furthercomprise a third capture zone intermediate the first and second capturezones, wherein the third capture zone comprises a paclitaxel materialcapable of binding the detection reagent. In these embodiments,determining the quantity of paclitaxel can be determined by quantitatingthe amount of detection reagent bound to the third capture reagent.Quantitating the amount of detection reagent bound to the third capturereagent can include optical density measurement.

It will be appreciated that the method of the invention is applicable toother solid phase assays, including, for example, surface plasmonresonance (SPR) assays.

As noted above, more than one antibody, fragment, or derivative thereofcan be used in the method. In these embodiments, the first antibody canhave a relatively high K_(on) (e.g., greater than 1×10⁴) and the secondantibody can have a relatively low K_(off) (e.g., less than 1×10⁻³).

Furthermore, in certain embodiments, the capture zone can includemultiple capture positions (e.g., two or three lines, T1, T2, T3) toprovide multiple readings on the same sample allowing for increasereproducibility and expanded dynamic range.

In another aspect, the invention provides methods for therapeutic drugmonitored (TDM) guided paclitaxel therapy. In one embodiment, theinvention provides a method for monitoring the efficacy of a paclitaxeltherapy in a patient diagnosed with cancer, comprising:

(a) treating a cancer patient with paclitaxel at a first point in time;

(b) determining a first concentration of paclitaxel in the patient at afirst point in time, wherein determining the concentration comprises themethod noted above for assaying paclitaxel;

(c) treating the patient with paclitaxel at a second point in time;

(d) determining a second concentration of paclitaxel drug in the patientat a second point in time, wherein determining the concentrationcomprises the method noted above for assaying paclitaxel; and

(e) comparing the first and second concentrations of paclitaxel in thepatient to determine the efficacy of the cancer treatment.

In a related embodiment, the invention provides a method for PK-guideddosing of paclitaxel therapy in a patient diagnosed with cancer,comprising:

(a) treating the cancer patient with paclitaxel at a first point intime;

determining one or more pharmacokinetic parameters of paclitaxel in thepatient at a first point in time, wherein determining the one or morepharmacokinetic parameters comprises the method noted above for assayingpaclitaxel;

(b) treating the patient with paclitaxel at a second point in time usingthe PK information from first dosing;

(c) determining one or more pharmacokinetic parameters of paclitaxel inthe patient at a second point in time, wherein determining the one ormore pharmacokinetic parameters comprises the method noted above forassaying paclitaxel; and

(d) comparing one or more pharmacokinetic parameters of paclitaxel inthe subject at the first point in time with the levels at the secondpoint in time to confirm that appropriate dosing was achieved.

In certain embodiments, the pharmacokinetic parameters are selected fromthe group consisting of time to maximum concentration (T_(max)),concentration maximum (C_(max)), area under the curve (AUC), clearance(CL), volume of distribution (V_(d)), apparent volume of distributionduring the terminal phase (V_(Z)), apparent volume of distributionduring steady state (V_(ss)) and combinations thereof.

In further aspects, the invention provides devices and methods, similarto those described above for paclitaxel, but that are useful forassaying other therapeutic agents.

In one embodiment, the invention provides a lateral flow device,comprising:

(a) a sample receiving zone for receiving a liquid sample;

(b) a detection reagent zone in liquid communication with the samplereceiving zone and downstream in flow direction from the samplereceiving zone,

wherein the detection reagent zone comprises one or more detectionreagents deposited thereon;

(c) a capture zone in liquid communication with the detection reagentzone and downstream in flow direction from the detection reagent zone,

wherein the capture zone comprises one or more capture reagentsimmobilized thereon; and

(d) an absorbent zone in liquid communication with the capture zone anddownstream in flow direction from the capture reagent zone.

As noted above, more than one antibody, fragment, or derivative thereofcan be used in the method. In these embodiments, the first antibody canhave a relatively high K_(on) (e.g., greater than 1×10⁴) and the secondantibody can have a relatively low K_(off) (e.g., less than 1×10⁻³). Inone embodiment, the detection reagent comprises a first antibody havinga first affinity for the therapeutic drug and a second antibody having asecond affinity for the therapeutic drug, wherein the first affinity isgreater than the second affinity. Furthermore, in certain embodiments,the capture zone can include multiple capture positions (e.g., two orthree lines, T1, T2, T3) to provide multiple readings on the same sampleallowing for increase reproducibility and expanded dynamic range.

In the method, the detection reagent is an antibody labeled with adetectable reporting group, and the capture reagent is an antigen thatcompetes with the therapeutic drug for binding to the detection reagentor the capture reagent is an antibody capable of binding the detectionreagent. In certain embodiments, the capture reagents comprise a firstcapture reagent that is an antigen that competes with the therapeuticdrug for binding to the detection reagent, and a second capture reagentthat is an antibody capable of binding the detection reagent. The firstcapture reagent is immobilized upstream in flow direction from thesecond capture reagent. The distance between the sample receiving zoneand the first capture reagent can be varied to optimize detection of thetherapeutic drug.

In certain embodiments, the device further includes a third capturereagent immobilized intermediate the first and second capture reagents.In this embodiment, the third capture reagent is an antigen thatcompetes with the therapeutic drug for binding to the detection reagent.

In certain embodiments, the therapeutic agent is paclitaxel and thedetection reagent comprises a monoclonal antibody selected from 8A10 and3C6, or fragment or derivative thereof, wherein the antibody, orfragment or derivative binds to paclitaxel.

In another embodiment, the invention provides a method for assaying atherapeutic drug in a sample, comprising:

(a) applying a sample to a sample receiving zone of the device notedabove; and

(b) observing the amount of detection reagent bound to the one or moreimmobilized capture reagents.

The method can further include determining the quantity of therapeuticdrug in the sample by quantitating the amount of detection reagent boundto the one or more immobilized capture reagents (e.g., detection reagentis bound to a capture reagent in a first capture position, detectionreagent is bound to a capture reagent in a second capture position,detection reagent is bound to capture reagents in first and secondcapture positions, detection reagent is bound to capture reagents infirst, second, and third capture positions).

In further embodiments, TDM guided therapy methods are provided.

In one embodiment, the invention provides a method for monitoring theefficacy of a therapeutic treatment in a patient diagnosed with adisease or condition, comprising:

(a) treating a patient with a therapeutic agent at a first point intime;

(b) determining a first concentration of the therapeutic agent in thepatient at a first point in time, wherein determining the concentrationcomprises the methods described herein;

(c) treating the patient with the therapeutic agent at a second point intime;

(d) determining a second concentration of the therapeutic agent in thepatient at a second point in time, wherein determining the concentrationcomprises the methods described herein; and

(e) comparing the first and second concentrations in the patient todetermine the efficacy of the therapeutic treatment.

In another embodiment, the invention provides a method for PK-guideddosing of a therapeutic treatment in a patient diagnosed with a diseaseor condition, comprising:

(a) treating the patient with a therapeutic agent at a first point intime using the PK information from first dosing;

(b) determining one or more pharmacokinetic parameters of thetherapeutic agent in the patient at a first point in time, whereindetermining the one or more pharmacokinetic parameters comprisesassaying the therapeutic agent according to methods described herein;

(c) treating the patient with the therapeutic agent at a second point intime;

(d) determining one or more pharmacokinetic parameters of thetherapeutic agent in the patient at a second point in time, whereindetermining the one or more pharmacokinetic parameters comprisesassaying the therapeutic agent according to the methods describedherein; and

(e) comparing one or more pharmacokinetic parameters of the therapeuticagent in the subject at the first point in time with the levels at thesecond point in time to confirm that appropriate dosing was achieved.

In certain embodiments, the pharmacokinetic parameters are one or moreof time to maximum concentration (T_(max)), concentration maximum(C_(max)), area under the curve (AUC), clearance (CL), volume ofdistribution (V_(d)), apparent volume of distribution during theterminal phase (Vz), apparent volume of distribution during steady state(V_(ss)) and combinations thereof.

The above methods are applicable to disease or condition treatments thatbenefit from therapeutic drug monitoring. Representative diseases orconditions include cancer, inflammation, hypertension, cardiovascular,and pain. Representative therapeutic agents include paclitaxel,docetaxel, 7-epipaclitaxel, t-acetyl paclitaxel,10-desacetyl-paclitaxel, 10-desacetyl-7-epipaclitaxel,7-xylosylpaclitaxel, 10-desacetyl-7-glutarylpaclitaxel,7-N,N-dimethylglycylpaclitaxel, and 7-L-alanylpaclitaxel.

In a further aspect, the invention provides paclitaxel antibodies. Inone embodiment, the invention provides a monoclonal antibody selectedfrom 8A10 and 3C6, or fragment or derivative thereof, wherein theantibody, antibody fragment, or antibody derivative binds to paclitaxel.In certain embodiments, the monoclonal antibody, antibody fragment, orantibody derivative comprises one or more complementary determiningregions (CDRs) with at least 95% homology to a CDR contained in SEQ IDNOS: 2, 4, 6, or 8.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings.

FIG. 1 is a schematic illustration of pharmacokinetic-guided dosingconcept and process using a representative system and method of theinvention.

FIG. 2A is an illustration of a representative work flow for atherapeutic drug monitoring device in accordance with the invention.

FIG. 2B is an illustration of a representative test strip for lateralflow immunoassay in accordance with the invention.

FIG. 2C is an illustration of a representative test strip for apaclitaxel lateral flow immunoassay in accordance with the invention.

FIG. 3 is an image of representative test strips subject to assayconditions in accordance with the present invention in which the amountof a representative analyte (paclitaxel) was varied. In this assayconfiguration, the test strip includes three capture zones: Test Line 1and Test Line 2 show capture using immobilized antigen (paclitaxel, inthe form of immobilized BSA-paclitaxel) as the first capture material;Control Line shows capture using an immobilized antibody (goatanti-mouse antibody, GAM) as the second capture material. In this assaythe detection reagent was an anti-paclitaxel antibody (8A10) labeledwith colloidal gold.

FIG. 4 is a graph illustrating direct binding of intact IgGs (8A10 and3C6) of the invention to a BSA-paclitaxel antigen on an ELISA.

FIGS. 5A and 5B illustrate curves for 8A10 bound at lines T1 and T2 in arepresentative lateral flow assay of the invention carried out using adevice as schematically illustrated in FIG. 2C and as described inExample 3. FIG. 5A illustrates the standard curve, i.e., the ratio oftest line over control line (T/C) vs. paclitaxel concentration. Thelarge difference in ratio for 8A10 at T1 versus T2 for the lowerconcentrations indicates a much higher sensitivity for this class ofantibody when placed closer to the sample port, where concentration ofanalyte is likely to be higher. FIG. 5B illustrates the output intensityvs. position readout of scanned test strips as provided by a readerdevice.

FIGS. 6A and 6B illustrate curves for 3C6 bound at lines T1 and T2 in arepresentative lateral flow assay of the invention carried out using adevice as schematically illustrated in FIG. 2C and as described inExample 3. FIG. 6A illustrates the standard curve, i.e., the ratio oftest line over control line (T/C) vs. paclitaxel concentration. Therelatively minor difference in ratio for 3C6 at T1 versus T2 for thelower concentrations indicates a relatively low improvement insensitivity for this class of antibody would be gained for placing theantibody closer to the sample port, where concentration of analyte islikely to be higher. This class of antibody is characterized by locationindependent signal from the sample port. FIG. 6B illustrates the outputintensity vs. position readout of scanned test strips as provided by areader device.

FIGS. 7A and 7B illustrate curves for combined 8A10 and 3C6 bound atlines T1 and T2 in a representative lateral flow assay of the inventioncarried out using a device as schematically illustrated in FIG. 2C andas described in Example 3. FIG. 7A illustrates the standard curve, i.e.,the ratio of test line over control line (T/C) vs. paclitaxelconcentration. The high sensitivity of T1 and T2 was obtained bycombining the two classes of antibody (8A10 and 3C6) in the conjugatepad. This improved the dynamic range of the assay. FIG. 7B illustratesthe output intensity vs. position readout of scanned test strips asprovided by reader device.

DETAILED DESCRIPTION OF THE INVENTION

Today there are many therapeutic agents either in clinical use or indevelopment. The difficulty is not in finding drugs for treatment, butin tailoring treatments to fit patients. Personalized medicine withoutregard to pharmacokinetic (PK) variability will result inmisclassification of some patients due to either too much drug exposureresulting in toxicity among patients that would have benefitted fromlower amounts of administered drug or too little drug exposure insupposedly sensitive populations. Personalized medicine with therapeuticdrug monitoring (TDM) removes the PK variability and allows for correctclassification of the patients according to their biomarker.

The present invention provides a point-of-care (POC) therapeutic drugmonitoring (TDM) methods, devices, and related compositions forpharmacokinetic (PK)-guided dosing of therapeutic drugs.

FIG. 1 illustrates system characteristics, advantages and potentialconnectivity of the device with the hospital information system and/orlaboratory information system and enabling the data transfer to thephysician, ultimately supporting the drug dosage decision. The methodsand devices of the invention collect the patient's therapeutic drug(e.g., paclitaxel) PK data by taking the finger-prick blood samples overa period of 24 to 48 hours and the PK data is accessed by the physicianwho can analyze the data and determine whether the therapeutic drug doseshould be modified.

In one aspect, the invention provides methods and devices forimmunoassay in general, and methods and devices for immunoassay ofpaclitaxel in particular. Sometimes a low level of paclitaxel in thebiological sample is indicative of a need to increase dosage ofpaclitaxel to the subject, and a high level of paclitaxel in thebiological sample is indicative of a need to decrease the dosage ofpaclitaxel to the subject. The methods and devices of the inventionprovide information useful for making adjustments to the therapeuticregime for the subject.

The assay methods and devices provided herein are described in thecontext of compositions, methods, and devices for the detection andmonitoring of paclitaxel. However, it is appreciated that the format ofthe described compositions, methods, and devices are not so limited, andare readily applied more generally to monitoring any analyte of choice.

Representative Assay Methods and Devices

The present invention provides assay methods and devices for detectingor quantifying analytes (e.g., paclitaxel) in a sample.

The methods and devices can be used to assay a biological sample, suchas a sample obtained from a subject (patient) that has received atherapeutic agent (e.g., paclitaxel) for the treatment of a condition.The sample used in the assay is ultimately a liquid sample (e.g., blood,plasma, urine).

The methods of the invention are solid phase assays and therefore aresuited for adaptation to other solid phase assay configurations. Toexemplify the invention, the methods and devices are described using alateral flow assay configuration. It will be appreciated that othersolid phase assays know in the art can be configured in accordance withthe present methods and devices.

Lateral flow assay methods and devices can be used in accordance withthe present invention. Depending on the format of the lateral flow assaymethod and device, the assay reagents can be disposed in certainconfigurations. In such an embodiment, one reagent will act as a“detection reagent” and another reagent will act as a “capture reagent.”Within this format, the detection reagent is generally deposited on theconjugate pad at a location between the sample port and a location wherethe capture reagent is deposited. The detection reagent generallycomprises a detectable label, whereas the capture reagent is immobilizedin its location on the pad. Thus, during operation, a liquid sampleintroduced in the sample port can flow along the pad. The sample willcome into contact with the detection reagent first, and thensubsequently flow over the capture reagent.

A representative device for performing a lateral flow assay inaccordance with the invention is illustrated in FIG. 2A. Referring toFIG. 2A, device 100 is a cassette that includes housing 110 havingsample port 120, reading window 130, and test strip 200 (see FIG. 2B).In operation, a liquid sample to be analyzed is introduced to the teststrip through port 120 and is flowed along the test strip as indicatedby the flow direction (from sample pad 210 to absorbent pad 240. Thetest results can be viewed by observing the test strip through readingwindow 130.

The test strip includes several zones and reagents for carrying out theassay. Referring to FIGS. 2A and 2B, representative test strip 200includes sample pad 210, conjugate pad 220, membrane 230, and absorbentpad 240. Sample pad 210, conjugate pad 220, membrane 230, and absorbentpad 240 are in liquid communication such that liquid sample introducedto the sample pad flows through or across the conjugate pad and membraneto the absorbent pad. The size and configuration of the test stripcomponents can be varied to suit the particular assay to be performed.For example, one or more of the component pads and membrane can overlapto facilitate optimal flow from one component to the next (sample pad210 can overlap with conjugate pad 220, which may overlap with membrane230, which may overlap with absorbent pad 240, as shown in FIG. 2A). Thenature of the test strip zones is not particularly critical andmaterials for these components are known in the art.

The operation of the representative device is described as follows.Sample pad 210 receives the liquid sample to be tested. Sample flowsfrom sample pad to conjugate pad 220.

Conjugate pad 220 includes one or more detection reagents (e.g.,antibodies having an affinity for the analyte in the sample to beassayed and that are labeled to facilitate detection of the antibody inthe assay).

In certain embodiments, a single detection reagent is deposited on theconjugate pad. In other embodiments, two or more detection reagents(e.g., two different antibodies, such as first and second antibodieshaving different affinities for the analyte to be assayed, differentK_(on) rates, and/or different K_(off) rates) are deposited on theconjugate pad. The first and second affinities are not the same. In oneembodiment, the first K_(on) is greater than the second K_(on). Inanother embodiment, the second K_(off) is greater than the firstK_(off). The description and specification of antibody affinity, K_(on),and K_(off) rates described below in the context of the paclitaxel assayare applicable to the assay of therapeutic agents in general. The amountof first and second antibody deposited can be varied and need not be thesame.

The detection reagent(s) deposited on conjugate pad 220 are mobilized bythe liquid sample and flow with the sample to membrane 230. When analyteis present in the sample, binding between the analyte and detectionreagent begins to occur once the sample contacts the detection reagents.Capture of the detection reagents, some of which may include boundanalyte and some of which may not, occurs on membrane 230.

Membrane 230 includes at least two capture zones: a first capture zonefor capturing detection reagent that does not include bound analyte(test line) (see 232 in FIGS. 2A, 2B, and 2C) and a second capture zonefor capturing excess detection reagent that does include bound analyte(control line) (see 238 in FIGS. 2A, 2B, and 2C). The first capture zoneincludes a first capture material (e.g., an immobilized antigen) that iseffective for capturing the detection reagent that does not includebound analyte (i.e., free detection reagent). The second capture zoneincludes a second capture material (e.g., an immobilized antibody) thatis effective for capturing the detection reagent with or without boundanalyte. The amount of detection reagent captured by the first andsecond capture materials, respectively, will depend on the amount ofanalyte present in the sample. The assay described above is acompetitive assay in which the analyte and first capture materialcompete for affinity binding to the detection reagent. The greater theamount of analyte present in the sample, the lesser the amount ofdetection reagent captured by the first capture material. Due todepletion of capture material, the lesser the amount of the analytepresent in the sample, the more detection reagent being capture by thefirst capture material and therefore less available for capture by thesecond capture material. The ratio of the intensity of the first andsecond capture lines give the best value for quantitation of theanalyte.

In certain embodiments, the capture zone includes two or more firstcapture zones (e.g., 232 and 234 in FIGS. 2B and 2C) for capturingdetection reagent that does not include bound analyte. In certainembodiments, the capture zone includes two or more second capture zones(e.g., 236 and 238 in FIG. 2B) for capturing detection reagent.

The illustrated approach of the lateral flow cassette can utilize anycompatible reader with the appropriate sensitivity for detection ofsignal from the flow cassette and the ability to calibrate and quantifysuch a signal. Beneficial features of any reader can include ease of usefeatures, including touch screen, integrated RFID or integrated barcodereader, and the capacity to easily export results, such as to a memorycard or USB stick. The reader preferably has pre-installed softwarefacilitating an interface in a selection of languages. The readerpreferably has a high memory capacity to facilitate storage of multiple(such as >1000) results and can save >100 distinct test methodprotocols. The reader can contain connectivity to facilitate itsintegration into a larger system, such as through LAN or WLANconnectivity to LIS or cloud based data storage and management systems.Finally, multiple USB ports are desirable for additional connectivitycapacities, such as to facilitate connection to external printers, andthe like.

A representative reader is the Qiagen's Reader ESEQuant LFR(commercially available from Qiagen, Germany), which has beendemonstrated as a compatible effective reader for the inclusion of thelateral flow cassette described herein. This reader is a small, portabledevice with internal rechargeable battery allowing it to operate out inthe field and serves the requirements of the point-of-care (POC) device.The lateral flow cassette is scanned using a confocal camera systemembedded in the reader. On board image analysis system is fullyfunctional with the bar code reader of the lateral flow cassettes sothat analysis method can be easily uploaded to the device.

Detection Reagents. In certain embodiments, the detection reagent is atleast one antibody, antibody fragment, or antibody derivative, asdescribed herein. The detection reagent is capable of binding theanalyte in the sample (e.g., paclitaxel) and when the detection reagentdoes not bind paclitaxel in the sample, the detection reagent binds tothe capture reagent.

The detection reagents include a moiety or label that can provide adetectable signal capable of reliable quantification. Suitable moietiesinclude those known in the immunoassay art that provide colorimetric,fluorescent, chemiluminescent, enzymatic, or radiometric signals.Representative moieties include that those provide a detectable signalthat is visual and may not require instrumentation to read (e.g.,colored moieties or enzymes that generate colored moieties or enzymatic.Quantitation is typically achieved through instrumental analysis of thedetectable signal. In one embodiment, the detection reagent is anantibody labeled with colloidal gold, which can be visually observed.

Gold colloids are generated from reduction of gold chloride with amonodisperse nature, which are of a controlled and uniform diameter,such as 40 nm monodisperse colloid. An antibody is conjugated withcolloidal gold through passive absorption.

As noted above, in preferred embodiments, multiple (i.e., more than onetype of) antibodies, antibody fragments, or antibody derivatives areused. In some embodiments, the multiple (distinct) antibodies, antibodyfragments, or antibody derivatives are combined and deposited in thesame location on the test strip (i.e., conjugate pad).

Two distinct anti-paclitaxel antibodies are described herein, 3C6 and8A10. The 3C6 antibody is highly specific for paclitaxel, whereas the8A10 antibody is less specific for paclitaxel and has a broader affinityto taxanes in general. Although, the two antibodies behave similarly intraditional competitive ELISA, it was surprisingly found that in solidphase lateral flow assays, the signal provided by 8A10 was improved bymoving the first capture reagent (e.g., T1 location) closer to thesample port, as compared to 3C6, which was independent of location (T1or T2). T1 being close to the sample application is exposed to higherconcentration of the analyte, and T2 being further from the sampleapplication is exposed to lower concentration of the analyte. This is asurprising finding that optimal placement of the capture line(s) isrelated to the Kon and Koff values of the antibodies used in the method.The availability of 3C6 allows for construction of multiple line deviceswherein the high Kon antibody (e.g., 8A10) is deposited as close to thesample origin as possible and the low Koff antibody (e.g., 3C6) isdeposited along the pad to provide a second/third/fourth, etc., readout.

Accordingly, various modifications can be made to the lateral flowcassette device to facilitate or confer various detection properties.For example, to expand the dynamic range of the device, multiple testlines (T1, T2, etc.) with the use of multiple affinity antibodies, thedynamic range and/or the reproducibility of the assay can be expanded.The description and specification of positioning capture reagents (T/C)on the test strip described below in the context of the representativepaclitaxel assay is applicable to positioning of capture reagents inassay of the invention in general.

The preparation of representative detection reagents (e.g., paclitaxelantibody-colloidal gold conjugate) useful in the assays of the inventionare described in Example 1.

Capture Reagents. The capture reagents serve to capture the detectionreagent allowing for observation and quantitation of a detectable signalin the assay. As noted above, the assay methods and devices includefirst and second capture materials immobilized at first and secondcapture zones, respectively.

In one embodiment, the capture reagent is an immobilized analyte (e.g.,paclitaxel complex), which is an immobilized antigen when the detectionreagent is an antibody, that captures detection reagent that does notinclude bound analyte. The immobilized analyte can be directlyimmobilized to the test strip. Alternatively, the immobilized analytecan be immobilized via a linker or carrier material (e.g., analyteconjugated to a carrier protein, such as albumin). In such anembodiment, the capture reagent is the first capture material asdescribed above.

In one embodiment, the capture reagent is an immobilized antibody thatcaptures detection reagent that captures detection reagent with orwithout bound analyte. In embodiments in which the detection reagent isa mouse monoclonal antibody, the capture reagent is an anti-mouseantibody (e.g., goat anti-mouse antibody, GAM antibody). In such anembodiment, the capture reagent is the second capture material asdescribed above.

The preparation of representative capture reagents (e.g.,BSA-paclitaxel) useful in the assays of the invention are described inExample 1.

Alternative Assay Configurations. The lateral flow assay of theinvention described herein is a solid phase immunoassay. It will beappreciated that the format of the assay and device can be inverted fromthe format described above such that the detection reagent is thelabeled antigen (e.g., BSA-paclitaxel with detectable label) and thecapture reagent is the one or more antibody, antibody fragment, orantibody derivative (i.e., immobilized in the capture zone). In theoperation of such a format, the sample flows through/across thedeposited labeled antigen and subsequently contacts the immobilizedantibody, antibody fragment, or antibody derivative. At that point, thefree analyte (e.g., paclitaxel) initially present in the sample competeswith the labeled antigen for binding to the immobilized antibody,antibody fragment, or antibody derivative. As above, the device caninclude multiple, distinct antibodies, antibody fragments, or antibodyderivatives immobilized at the same or different locations. The capturereagent can be at the same or different locations. In all embodimentswhere the test strip has multiple locations where capture reagent isimmobilized, an appropriate reader is used that can detect signal inthose locations.

It is noted that the present devices, systems, compositions, and methodsare generally described herein in terms of a lateral flow assay.However, the general strategy for monitoring an antigen of choice, asdescribed herein, does not need to be limited to lateral flow assayformats, but can applied to other assay formats, such as other solidphase immunoassays (surface plasmon resonance assays), which aregenerally well-known in the art. Accordingly, notwithstandingdescription addressing lateral flow format, the present disclosure alsoencompasses devices, systems, compositions, and methods that incorporateany known assay format. In some embodiments, the assay format includesimmobilization of capture reagents, such as the antigen conjugate (e.g.,paclitaxel conjugate) or antigen binding reagents (e.g., anti-paclitaxelantibodies, fragments, derivatives) on a substrate. The substrate can beany known appropriate substrate for an assay format, such asnitrocellulose or glass. In some embodiments, the substrate is ananostructure. In some embodiments, the substrate can comprise orconsist of carbon nanostructures, such as carbon nanotubes, to which thecapture reagents can be immobilized.

Representative Paclitaxel Assay. FIG. 2C is an illustration of arepresentative test strip for a paclitaxel lateral flow immunoassay inaccordance with the invention. Referring to FIG. 2C, representative teststrip 200 includes sample pad 210, conjugate pad 220, membrane 230 withfirst capture zones 232 and 234 (T1 and T2) and second capture zone 238(C), and absorbent pad 240. As noted above with regard to FIGS. 2A and2B, sample pad 210, conjugate pad 220, membrane 230, and absorbent pad240 are in liquid communication such that liquid sample introduced tothe sample pad flows through or across the conjugate pad and membrane tothe absorbent pad; the size and configuration of the test stripcomponents can be varied to suit the paclitaxel assay to be performed(e.g., one or more of the component pads and membrane can overlap tofacilitate optimal flow from one component to the next, as shown in FIG.2A).

In one embodiment, the invention provides a method for assayingpaclitaxel in a liquid sample, comprising

(a) applying a liquid sample comprising paclitaxel to a lateral flowassay device, the device having

(i) a sample receiving zone for receiving the liquid sample;

(ii) a detection reagent zone in liquid communication with the samplereceiving zone and downstream in flow direction from the samplereceiving zone,

wherein the detection reagent zone comprises a detection reagentdeposited thereon,

wherein the detection reagent is a paclitaxel antibody, or fragment orderivative thereof that binds paclitaxel, labeled with a detectablereporting group; and

(iii) a capture zone in liquid communication with the detection reagentzone and downstream in flow direction from the detection reagent zone,

wherein the capture zone comprises first and second capture reagentsimmobilized thereon, the first capture reagent positioned upstream inflow direction from the second capture reagent,

wherein the first capture reagent is a paclitaxel material capable ofbinding the detection reagent, and

wherein the second capture reagent is an antibody capable of binding thedetection reagent;

(b) allowing the sample to flow from the sample receiving zone throughthe detection reagent zone to provide a detection reagent withpaclitaxel (e.g., combination of detection agent with bound paclitaxel,optionally free detection reagent, and optionally free paclitaxel);

(c) allowing the detection reagent with paclitaxel to flow through thecapture zone,

whereby the first capture reagent binds free detection reagent toprovide detection reagent bound to the first capture reagent, and

whereby the second capture reagent binds detection reagent with orwithout bound paclitaxel; and

(d) observing the amount of detection reagent bound to the first capturereagent relative to the second capture reagent.

In certain embodiments, the method further comprises determining thequantity of paclitaxel in the sample by quantitating the amount ofdetection reagent bound to the first capture reagent. Quantitating theamount of detection reagent bound to the first capture reagent includesoptical density measurements, among others.

Suitable detectable reporting groups are described above. In oneembodiment, the detectable reporting group is colloidal gold.

The paclitaxel antibody, or fragment or derivative thereof, useful inthe present methods have a K_(on) greater than about 1×10⁴.Representative K_(on) values are greater than about 2×10⁴, 4×10⁴, 8×10⁴,1×10⁵, 1×10⁶, and 1×10⁷). Preferred ranges are from about 1×10⁴ to about1×10⁷.

The paclitaxel antibody, or fragment or derivative thereof, useful inthe present methods have a K_(off) less than about 1×10⁻³.Representative K_(off) values are less than about less than about1×10⁻³, 1×10⁻⁴, 1×10⁻⁵, and 1×10⁻⁷. Preferred K_(off) values range fromabout 1×10⁻³ to 1×10⁻⁷.

In certain embodiments, the paclitaxel antibody, or fragment orderivative thereof, has a K_(on) from about 1×10⁴ to about 1×10⁶ and aK_(off) from about 1×10⁻³ to about 1×10⁻⁴.

Methods for determining the K_(on) values and K_(off) values aredescribed in Example 4.

In one embodiment, the antibody has a high K_(on) and low K_(off) (e.g.,minimum K_(on) is 2.0×10⁵ and maximum K_(off) is 1.0×10⁻³). In thisembodiment, the capture line is placed at 0.0 to 0.4 T/C. For thisclass, monoclonal antibody engineering would focus on keeping K_(off)constant while increasing K_(on) as much as possible. The greater theK_(on) the better is the antibody detection.

In another embodiment, the antibody has a low K_(on) and high K_(off)(e.g., minimum K_(on) is 2.0×10⁴ and maximum K_(off) is 2.0×10⁻⁴. Inthis embodiment, the capture line is placed at 0.2-1.0 T/C. For thisclass, monoclonal antibody engineering would focus on keeping K_(on)constant while decreasing K_(off) as much as possible. The lower the offrate the better is the antibody for detection.

In certain embodiments, the paclitaxel antibody is 3C6. In otherembodiments, the paclitaxel antibody is 8A10. In further embodiments,combinations of paclitaxel antibodies can be used (e.g., 3C6 and 8A10).These antibodies are described in detail below.

In the assay, the first capture zone includes an immobilized paclitaxelmaterial that serves is a paclitaxel antigen that competes withpaclitaxel for binding to the detection reagent. The first capture zonecaptures detection reagent that does not include bound paclitaxel (i.e.,free detection reagent). In certain embodiments, the paclitaxel materialis a paclitaxel protein conjugate. Suitable protein conjugates includeserum albumin conjugates, such as BSA-paclitaxel.

In the assay, the second capture zone includes an immobilized antibodycapable of binding the detection reagent. In certain embodiments, theantibody is a goat anti-mouse antibody.

As noted above, the paclitaxel detection sensitivity in the assay can beoptimized by varying the distance between the point at which the sampleis introduced to the lateral flow device (e.g., sample receiving zone)and the first capture reagent. In certain embodiments, the distancebetween the sample receiving zone and the first capture reagent isminimized to optimize paclitaxel detection sensitivity. In certainembodiments, the distance is less than 20 mm, less than 10 mm, less than5 mm, less than 3 mm, less than 2 mm, or less than 1 mm. In certainembodiments, the distance is from 20 to 1 mm, 10 to 1 mm, 5 to 1 mm, 3to 1 mm, or 2 to 1 mm.

The optimization can be described as relative positioning of T (testline) and C (control line): T/C, which is defined as the distance fromorigin to T/distance from origin to C ratio, where the origin is definedas the upstream edge of the capture zone (upstream edge of membrane 230in FIGS. 2A-2C). T/C can be greater than about 0.0 (i.e., first capturereagent is located at upstream edge of capture zone), or about 0.01,about 0.02, about 0.04, about 0.08, about 0.10, about 0.20, about 0.40,about 0.80, or less than about 1.0 (i.e., first capture reagent islocated at the downstream edge of the capture zone, with second capturereagent located intermediate the first capture reagent and thedownstream edge of the capture zone). Preferably, T/C is from about 0.2to about 0.7.

In certain embodiments, the ratio of the first distance to the seconddistance is from about 0.0 to about 0.40. In other embodiments, theratio of the first distance to the second distance is from about 0.20 toabout 1.0.

In certain embodiments, the amount of excess detection reagent that isbound to the second capture reagent is observed and measured. In certainembodiments, determining the quantity of paclitaxel in the sample isdetermined by relating the final capture reagent (test line) to thesecond capture reagent (control line).

As noted above, representative assay of the invention include more thanone first capture reagents in more than one first capture zone. Incertain of these embodiments, the method further includes a thirdcapture zone (see T2, 234 in FIG. 2C) intermediate the first (T1, 232 inFIG. 2C) and second (C, 238 in FIG. 2C) capture zones, wherein the thirdcapture zone comprises a paclitaxel material capable of binding thedetection reagent. The paclitaxel material in the first and third zonescan be the same or different. In certain of these embodiments, thequantity of paclitaxel in the sample is determined by quantitating theamount of detection reagent bound to the first and second capturereagents. Quantitating the amount of detection reagent bound to thefirst and second capture reagents can include optical densitymeasurement.

In certain embodiments of the method, the lateral flow device furthercomprises an absorbent zone in liquid communication with the capturereagent zone and downstream in flow direction from the capture reagentzone.

FIG. 3 is an image of representative test strips subject to assayconditions in which the amount of a representative analyte (paclitaxel)was varied. In these assay configurations, membrane 230 includes threecapture zones: Test Line 1 and Test Line 2 show capture usingimmobilized antigen (paclitaxel, in the form of immobilizedBSA-paclitaxel) as the first capture material; Control Line showscapture using an immobilized antibody (goat anti-mouse antibody) as thesecond capture material. In this assay the detection reagent was ananti-paclitaxel antibody (8A10) labeled with colloidal gold.

It is noted that the methods and devices of the invention are useful fordetecting levels of paclitaxel, including any formulation of paclitaxelor paclitaxel prodrug, in a biological sample. The formulations ofpaclitaxel include any known agents to facilitate delivery ofpaclitaxel, such as polymeric nanoparticles, lipid-based nanoparticleformulations, polymer conjugates, inorganic nanoparticles, carbonnanotubes, nanocrystals, and cyclodextrin nanoparticles.

The description of a representative lateral flow immunoassay inaccordance with the methods and devices of the invention is described inExample 3.

Paclitaxel Antibodies

In another aspect, the invention provides antibodies (e.g., monoclonalantibodies or mAbs) that bind paclitaxel. The mAbs, referred to as 8A10and 3C6, were purified from an antibody-rich harvested medium usingMabSelect (GE Healthcare, Pittsburgh, Pa.). The mAbs were selected basedon their binding to BSA-paclitaxel, which was prepared as described inJ-G Leu et al., Cancer Res. (1993) 53:1388-1391.

In one aspect, the invention provides a monoclonal antibody selectedfrom 8A10, 3C6, and fragments or derivatives thereof, wherein theantibody, antibody fragment, or antibody derivative binds paclitaxel.

As used herein, the term “antibody” encompasses antibodies and antibodyfragments thereof, derived from any antibody-producing mammal (e.g.,mouse, rat, rabbit, camelid, and primate, including human) orsynthetically or recombinantly produced, that specifically binds to atarget of interest (e.g., paclitaxel) or portions thereof. Exemplaryantibodies include polyclonal, monoclonal, and recombinant antibodies;multispecific antibodies (e.g., bispecific antibodies); humanizedantibodies; murine antibodies; chimeric, mouse-human, mouse-primate,primate-human monoclonal antibodies; and anti-idiotype antibodies, andmay be any intact molecule or fragment thereof, such as an antigenbinding fragment. As described herein, monoclonal antibodies arepreferable because they provide for increased specificity in binding ofthe antigen of choice, such as a therapeutic drug (e.g., paclitaxel).

As used herein, the term “antigen binding fragment” refers to theantigen binding or variable region from or related to a full-lengthantibody. Illustrative examples of antibody fragments include Fab, Fab′,F(ab)₂, F(ab′)₂, and Fv fragments, scFv fragments, diabodies,nanobodies, linear antibodies, single-chain antibody molecules, andmultispecific antibodies formed from antibody fragments.

As used herein, a “single-chain Fv” or “scFv” antibody fragmentcomprises the V_(H) and V_(L) domains of an antibody, wherein thesedomains are present in a single polypeptide chain. Generally, the Fvpolypeptide further comprises a polypeptide linker between the V_(H) andV_(L) domains, which enables the scFv to form the desired structure forantigen binding.

As used herein, a “chimeric antibody” is a recombinant protein thatcontains the variable domains and complementarity-determining regionsderived from a non-human species (e.g., rodent) antibody, while theremainder of the antibody molecule is derived from a human antibody.

As used herein, a “humanized antibody” is a chimeric antibody thatcomprises a minimal sequence that conforms to specificcomplementarity-determining regions derived from non-humanimmunoglobulin that is transplanted into a human antibody framework.Humanized antibodies are typically recombinant proteins in which onlythe antibody complementarity-determining regions are of non-humanorigin.

As used herein, the term “derivative” indicates that the antibody orantibody fragment has been produced from a reference antibody. Forexample, sometimes it is desirable to modify or enhance bindingcharacteristics of a reference antibody. Thus, the antibody can besubjected to various modifications, including mutations subjected to theencoding DNA, to alter binding properties. The resulting antibody withaltered properties is then referred to as a “derivative” of thereference antibody. For example, an antibody derivative can be anantibody that contains mutations resulting from affinity maturationprocesses that were applied to the reference antibody (or the nucleicacids encoding the reference antibody). Such mutations can result inantibodies with altered (e.g., improved) binding affinity, selectivity,and the like.

In some embodiments, the antibody, antibody fragment, or antibodyderivative comprises one or more complementary determining regions(CDRs) with at least 95% homology to a CDR contained in SEQ ID NOS: 2,4, 6, or 8. SEQ ID NOS: 2 and 4, described in more detail below, are theamino acid sequences of the variable region of the 8A10 light and heavychains, respectively. SEQ ID NOS: 6 and 8, also described in more detailbelow, are the amino acid sequences of the variable region of the 3C6light and heavy chains, respectively. Each variable region containsthree complementary determining regions (CDRs), which are indicatedbelow. In some embodiments, the antibody, antibody fragment, or antibodyderivative comprises one, two, or all three of complementary determiningregions (CDRs) with at least 95% homology to one, two, or all three ofthe CDRs contained in any one SEQ ID NOS: 2, 4, 6, or 8. In someembodiments, the antibody, antibody fragment, or antibody derivative hasa heavy and light chain, which comprise one, two, three, four, five, orall six, of the CDR regions provided by the 8A10 variable light andheavy regions (in SEQ ID NOS: 2 and 4). In some embodiments, theantibody, antibody fragment, or antibody derivative has a heavy andlight chain, which comprise one, two, three, four, five, or all six, ofthe CDR regions provided by the 3C6 variable light and heavy regions (inSEQ ID NOS:6 and 8).

In some embodiments, the one or more CDRs have a sequence identity withat least 95%, 96%, 97%, 98%, 99%, or 100% homology to one or more CDRsin any one SEQ ID NOS: 2, 4, 6, or 8. As used herein, the term “percenthomology” or “percent homologous,” when used in connection with apolypeptide used in the practice of the present invention, is defined asthe percentage of amino acid residues in a polypeptide sequence that arehomologous with the amino acid sequence of a specified polypeptide afteraligning the sequences to achieve the maximum percent homology. Whenmaking the comparison, no gaps are introduced into the biomarkersequences in order to achieve the best alignment. Amino acid sequencehomology can be determined, for example, in the following manner. Theamino acid sequence of a polypeptide is used to search a proteinsequence database, such as the GenBank database using the BLASTPprogram. The program is used in the ungapped mode. Default filtering isused to remove sequence homologies due to regions of low complexity. Thedefault parameters of BLASTP are utilized.

The production, processing, purification, characterization, andoptimization of representative paclitaxel antibodies (3C6 and 8A10)useful in the assay methods of the invention are described in Example 2.The antibodies of the invention were generated by immunizing mice witheither TAXOL® or baccatin conjugated to KLH. The antibodies aresummarized as: 3C6 anti TAXOL® IgG_(2a,k), 3H5 anti-baccatin III IgG₁,8A10 anti-taxane IgG_(2a). Cross-reactivity profile of 3C6, 3H5, and8A10 antibodies are shown by their IC₅₀ values towards various taxanesin Table 1.

TABLE 1 Paclitaxel Antibody Cross-Reactivity. Taxane 3C6 3H5 8A10paclitaxel (TAXOL ®) 10 nM >316 nM 7 nM 10-deacetyltaxol 15 nM >333 nM10 nM 7-epi-10-deacetyltaxol 25 nM >333 nM 15 nM7-xylosyl-10-deacetyltaxol 30 nM >286 nM 17 nM 7-epi-taxol 80 nM >316 nM50 nM cephalomannine 220 nM >325 nM 8 nM baccatin III >511 nM 10 nM 12nM baccatin V >460 nM 10 nM 10 nM 10-deacetylbaccatin III >551 nM 230 nM21 nM 7-epi-10-deacetylbaccatin III >469 nM 150 nM 27 nM TAXOTERE ® >318nM >318 nM 10 nM (docetaxel formulation) 2-debenzoyl-2-(p- >293 nM >293nM >293 nM trifluoromethylbenzoyl)taxol20-acetoxy-4-deacetyl-5-epi- >310 nM >310 nM >293 nM 20,O-secotaxol

In order to effectively utilize antibodies in diagnostic applications toachieve a dynamic detection range, the two key factors that need to besuccessfully optimized are the antibodies' specificity and affinitytoward the targeted antigen. The 3C6 and 8A10 monoclonal antibodies havehigh specificity and affinity to paclitaxel, which indicates that thesespecifically bind to a single epitope of the targeted paclitaxel antigenin a highly homogeneous manner. This specificity also helps eliminatecross reactivity problems in a detection assay. In conjunction withspecificity, monoclonal antibodies can also have optimized and improvedaffinities toward the antigen in order to achieve a dynamic detectionrange in diagnostic tests, which can be achieved by the process ofaffinity maturation. Additionally, the mAb can be selected for rapidK_(on) to be compatible with the rapid flow assay.

As illustrated in FIG. 4, both the 3C6 and 8A10 monoclonal antibodieshave apparent K_(d) values of about 10 nM with a sensitivity limit ofabout 100-200 ng/mL.

The following examples are provided for the purpose of illustrating, notlimiting, the invention.

EXAMPLE 1 Assay Reagents

In this example, the preparation of representative detection reagentsand capture reagents useful in the assay methods and devices of theinvention are described.

Detection reagents: antibody-colloidal gold conjugates. Briefly,antibodies (see Example 2) were diluted to 1 mg/mL in 0.5× PBS and thefollowing steps were taken: (1) shake or swirl gold to resuspend anysettled gold then place 0.5 mL Naked Gold sol into 10 clean individualtest tubes; (2) each tube was labeled with the pH value (or 1 through10) from the provided pH charts; (3) pH charts were used to add varyingamounts of buffer in microliters to each test tube, and shake to mix;(4) place each tube on a low speed vortexer and add the antibodysolution, and mix thoroughly (about 2 to 3 seconds), for the 20 nm gold,14 μL of a 2 mg/mL solution of antibody or protein is optimal; (5) adeepening purple color and/or black precipitate on some tubes indicatethat the antibody or protein is below its isoelectric point, leading tocross-linking of individual gold solutions (cross-linked solutionscannot be used in immunological assays are discarded; deep purplesolutions are mostly inactive as well; only tubes with a slight purplecolor or no change in color are useful for immunological assays; (6)allow the reaction to continue for a total of 30 minutes; and (7) stopthe reaction by the addition of 50 μL of blocking solution.

Capture reagents: paclitaxel-albumin conjugates. Paclitaxel-albuminconjugates (e.g., BSA-paclitaxel) were prepared as described in J-G Leuet al., Cancer Res. (1993) 53:1388-1391 was generally followed. Forsynthesis of 2′-hemisuccinitaxol, the crystals are the starting materialfor conjugation of the taxol to carrier protein. Taxol (20 mg) andsuccinic anhydride (36 mg) were dried for 4 h at room temperature undervacuum over P₂O₅ and dissolved in 480 μL of dry pyridine. After standingat room temperature overnight, the pyridine was removed under vacuum andthe residue was washed once with 2 mL of distilled water. Acetone (1 mL)was added, and distilled water was added dropwise to the acetonesolution until a few crystals (2′-hemisuccinyltaxol) appeared. Themixture was kept at 4° C. for 3 h and the crystals were recovered byfiltration and dried under vacuum. The product yield was 70%.

The dialysis step removes any unconjugated taxol. 2′-Hemisuccinyltaxol(10 mg) was dissolved in 1 mL DMSO and 300 μL acetonitrile, and 50 μL(35 mg; 0.19 mmol) of n-tributylamine was then added. The mixture wascooled to 4° C. in an ice bath, and 25 μL (25 mg; 0.18 mmol) ofisobutylchloroformate was added to the mixture which was kept on ice for30 min. The solution was added dropwise into a BSA solution [25 mg,(3.73×10⁻⁴ mmol) in 3 mL of distilled water, pH 9.5, at 4° C]. The pHwas adjusted immediately to 7.5 with IN HCl and the mixture was kept at4° C. overnight and dialyzed against PBS at 4° C. overnight.

In one exemplary embodiment, a lateral flow system was evaluated. A 0.5mg/mL BSA-paclitaxel (Test line) and 0.5 mg/mL goat anti-mouse antibody(Control line) were striped onto the system's membrane. Paclitaxelantibody-colloidal gold conjugate was flowed through the system. Theantibody-colloidal gold conjugate bound to BSA-paclitaxel immobilized onthe membrane and generated a strong signal. The signal was specific topaclitaxel because a decreased signal was observed when paclitaxel wasadded to the spiked into the samples.

EXAMPLE 2 Paclitaxel Antibodies

In this example, the production, processing, purification,characterization, and optimization of representative paclitaxelantibodies useful in the methods and devices of the invention aredescribed.

Antibody production and processing. Cells were grown in CCM1 (Hyclone)with 5-10% FBS and 1× Pen/Strep. Cells were split (1:4) once theyreached densities of >1×10⁶ cells/mL. Cells were then frozen and storedin 2 separate liquid nitrogen cryogenic tanks as backups. Cells werecultured in roller bottles until a density of 1×10⁶ cells/mL wasreached. At that point, cultures were no longer fed and cell viabilitywas monitored daily. Once cell viability decreased to <50%, cells wereremoved and the antibody-rich medium was harvested.

Affinity purification of antibody. Diafiltration was performed usingPBS, pH 7.4 and the harvested antibody was concentrated 10-fold using a50 Kd cut-off membrane. MabSelect (GE Healthcare, Pittsburgh, Pa.) wasused for affinity purification. The hydrophilic, high-flow agarose bead,optimized for both capacity and throughput, and the oriented coupling ofthe rProtein A ligand, deliver a product pool that is high in purity andyield.

Purification Run Program.

Column: XK 16/40 (16 mm i.d., 20 cm bed height).

Sample: Clarified hybridoma spent media.

Loading Buffer A: 20 mM NaH₂PO₄, 0.15 M NaCl, pH 7.2.

Elution Buffer B: 0.1 M Na3-citrate, pH 3.6.

Antibody affinity. A plot of the results of direct binding of intactIgGs (8A10 and 3C6) to BSA-paclitaxel antigen is shown in FIG. 4. 50 μLof BSA-paclitaxel antigen at a concentration of 3.5 μg/mL in 1× PBS wasbound to a plate and incubated at 4° C. overnight. The plate was washedfour ties with PBS/0.05% Tween 20. The plate was blocked for 2 hourswith 1% BSA in PBS/0.05% Tween 20 at room temperature. The plate waswashed four ties with PBS/0.05% Tween 20. 50 μL of antibody sample (8A10and 3C6) was bound (start at 300 nM and titer down by 3s). The plate waswashed four times with PBS/0.05% Tween 20. 50 μL of goat anti mIgGhorseradish peroxidase (1:5000 dilution) was bound and then 50 μL ofsubstrate solution TMB was added and the color allowed to develop. Thereaction was stopped with 50 μL 1M HCl and the optical density was readat 450 nm. The results of direct binding of intact IgGs (8A10 and 3C6)to BSA-paclitaxel antigen are shown in FIG. 4.

Antibody optimization. Antibody optimization was performed that includedaffinity maturation. This approach involves: (1) characterizing theantibodies by sequencing antibodies produced from hybridoma cell lines,(2) constructing antibody libraries focused on CDR regions; (3)screening for beneficial mutations, and (4) combining beneficialmutations.

Step 1: Cloning and Expression of Anti-Paclitaxel mAb as a Fab

A. Sequencing of Variable Regions of Antibodies (8A10 and 3C6) Producedfrom Hybridoma Cells.

i. Total RNA Extraction and mRNA Denaturation

Total RNA was extracted from the two hybridoma cells (8A10 and 3C6)using QIAGEN RNeasy Mini Kit. The mRNA mix (described below) wasincubated at 72° C. for 3 min, then cooled down to 42° C. for 2 min.After cooling, the tube was spun briefly for 10 seconds at 14,000×g tocollect the content at the bottom.

mRNA Mix RNA template (0.2-0.4 ug) 1-2.75 uL 5′-RAGE primer A 1 uLRNase-free water to 3.75 uL

ii. cDNA Synthesis and 5′ RAGE Reaction

cDNA synthesis and 5′ RAGE reaction were performed as shown below.

cDNA synthesis 5′ RAGE reaction SMARTer II A oligo 1 uL 2X PCR mix 10 uL5X First-strand buffer 2 uL cDNA 1 uL DTT (20 mM) 1 uL 10X UniversalPrimer mix 2 uL dNTP mix (10 mM) 1 uL Reverse primer 1 uL RNaseinhibitor 0.25 uL RNase-free water 6 uL SMARTScribe RT 1 uL mRNA mixafter dent 3.75 uL Total 10 uL 20 uL

iii. Analysis of PCR Reaction by Agarose Gel Electrophoresis

Products of PCR amplification were electrophoresed on an agarose gel toconfirm the presence of amplicons corresponding the light and heavychain variable regions of the 8A10 and 3C6 mAbs.

iv. Cloning, Sequencing and CDR Analysis

The PCR positive bands were cloned into a vector and sequenced. Antibodysequence analysis identified one light chain and one heavy chain for8A10 and 3C6. Initially for 3C6, only one light chain and no heavy chain(aberrant sequence) were identified. Therefore, a specific primer,designed according to the sequence obtained from the N-terminalsequencing results, was utilized to re-PCR the heavy chain.Consequently, identification of a heavy chain for 3C6 was accomplished.

8A10 Hybridoma

The 8A10 hybridoma sequencing results are set forth below.

The 8A10 Variable Light Chain nucleic acid sequence is SEQ ID NO: 1:

(SEQ ID NO: 1) GAC ATT GTG ATG ACC CAG TCT CAA AAA TTC ATG TCCATA ACA CTA GGA GAG AGG GTC AGC ATC ACC TGC AAGCCC AGT CAG AAT GTG GGT TCT GCT GTA ACC TGG TGGCAA CAG AAA CCA GGA CAA TCT CCT AAA CTA CTG ATTTAC TCA GCT TCC AAT CGG TAT ACT GGA GTC CCT GATCGC TTC ACA GGC AGT GGA TCT GGG ACA GAT TTC ACTCTC ACC ATT AGT AAT GTG CAG TCT GAA GAC CTG GCAGAT TAT TTC TGT CAA CAA TAT AGC AGC TAT CCG TACACG TTC GGA GGG GGG ACC AAG CTG GAA ATA AAA CG.

The sequences encoding the CDR regions are underlined.

The corresponding 8A10 Variable Light Chain amino acid sequence is SEQID NO: 2:

(SEQ ID NO: 2) DIVMTQSQKFMSITLGERVSITCKPSQNVGSAVTWWQQKPGQSPKLLIYSASNRYTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGG TKLEIKR.

The three CDR regions are underlined (i.e., CDRL1 is KPSQNVGSAVT (SEQ IDNO: 9), CDRL2 is SASNRYT (SEQ ID NO: 10), CDRL3 is QQYSSYPYT SEQ IDNO:11)).

The 8A10 Variable Heavy Chain nucleic acid sequence is SEQ ID NO: 3:

(SEQ ID NO: 3) GAG GTC CAG CTG CAA CAA TCT GGA CCT GAA CTG GTGAAG CCT GGG GCT TCA GTG AAG ATT TCC TGT AAG GCTTCT GGA TAC ACG TTC ACT GAC TCC ACC ATG AAC TGGGTG AAG CAG AGC CAT GGA AAG AGC CTT GAG TGG ATTGGA GAG ATT GAT CCT AAC AAT GGT GGT ACT AAC TACAAT CAG AAG TTC AAG GGC AAG GCC ACA TTG ACT GTAGAC AAG TCC TCC AGC ACA GCC TAT ATG GAG CTC CGCAGC CTG ACA TCT GAG GAC TCT GCA GTC TAT TAC TGTGCA AGA GGG GTC TGG GGC CAA GGC ACC ACT CTC ACA GTC TCC TCA.

The sequences encoding the CDR regions are underlined.

The corresponding 8A10 Variable Heavy Chain amino acid sequence is SEQID NO: 4:

(SEQ ID NO: 4) EVQLQQSGPELVKPGASVKISCKASGYTFTDSTMNWVKQSHGKSLEWIGEIDPNNGGTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARG VWGQGTTLTVSS.

The three CDR regions are underlined (i.e., CDRH1 is GYTFTDSTMN (SEQ IDNO: 12), CDRH2 is EIDPNNGGTNYNQKFKG (SEQ ID NO: 13), and CDRH3 is GV).

3C6 Hybridoma

The 3C6 hybridoma sequencing results are set forth below.

The 3C6 Variable Light Chain nucleic acid sequence is SEQ ID NO: 5:

(SEQ ID NO: 5) GAT GTT GTG ATG ACC CAA ACT CCA CTC TCC CTG CCTGTC AGT CTG GGA GAT CAA GCC TCC ATC TCT TGC AGATCT CGT CAG AGC CTT GTA CAC AGT AAT GGA AAC ACCTAT TTA CAT TGG TAC CTG CAG AAG CCA GGC CAG TCTCCA AAG CTC CTG ATC TAC AAA GTT TCC AAC CGA TTTTCT GGG GTC CCA GAC AGG TTC AGT GGT AGT GGA TCAGGG ACA GAA TTC ACA CTC GAG ATC AGC AGA GTG GAGGCT GAG GAT CTG GGA GTT TAT TTC TGC TCT CAA AGTACA CAT GTT CCT CCG ACG TTC GGT GGA GGC ACC AAG CTG GAA ATC AAA C.

The sequences encoding the CDR regions are underlined.

The corresponding 3C6 Variable Light Chain amino acid sequence is SEQ IDNO: 6:

(SEQ ID NO: 6) DVVMTQTPLSLPVSLGDQASISCRSRQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTEFTLEISRVEAEDLGVYFCSQSTHVP PTFGGGTKLEIK.

The three CDR regions are underlined (i.e., CDRL1 is RSRQSLVHSNGNTYLH(SEQ ID NO: 14, CDRL2 is KVSNRFS (SEQ ID NO: 15), and CDRL3 isSQSTHVPPT(SEQ id NO: 16)).

The 3C6 Variable Heavy Chain nucleic acid sequence is SEQ ID NO: 7:

(SEQ ID NO: 7) GAG GTG CAG CTT CAG GAG TCG GGA CCT AGT CTC GTGAAA CCT TCT CAG ACT CTG TCC CTC ACC TGT TCT GTCACT GGC GAC TCC ATC ACC AGT GGT TAC TGG AAC TGGATC CGG AAA TTC CCA GGG AAT AGA CTT GAG TAC ATGGGG TAC ATA AGC TAC AGT GGT AGC ACT TAC TAC AATCCG TCT CTC AAA AGT CGA ATC TCC ATC ACT CGA GACACA TCC AAG AAC CAG TAC TAC CTA CAT TTG ACT TCTGTG ACT ACT GAG GAC ACA GCC ACA TAT TAC TGT GCCCAA GGG GAT GGC GCC TAC TGG GGC CAA GGC ACC ACT CTC ACA GTC TCC TCA.

The sequences encoding the CDR regions are underlined.

The corresponding 3C6 Variable Heavy Chain amino acid sequence is SEQ IDNO: 8:

(SEQ ID NO: 8) EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKPGNRLEYMGYISYSGSTYYNPSLKSRISITRDTSKNQYYLHLTSVTTEDTATYYCAQGDGA YWGQGTTLTVSS.

The three CDR regions are underlined (i.e., CDRH1 is GDSITSGYWN (SEQ IDNO: 17), CDRH2 is YISYSGSTYYNPSLKS (SEQ ID NO: 18), and CDRH3 is GDGAY(SEQ ID NO: 19)).

TABLE 2 Comparison of N-terminal sequencing results with hybridomasequencing results for 8A10 and 3C6 mAbs. 8A10 3C6 Hybridoma Hybridomasequencing sequencing N-terminal data N-terminal data sequencingVariable Variable sequencing Variable Variable data Light Heavy dataLight Heavy Residue Calls Chain Chain Residue Calls Chain Chain 1 D, Q,F, (S, H, M) D 1 D, E D E 2 H, I, L, (V) I 2 V V V 3 V, S, Q, (T, K) V 3Q, (V) V Q 4 M, E, N, F M E 4 M, L M L 5 T, Q, V, (.R) T V 5 Q, T T Q 6Q, Q, N, Y Q Q 6 Q, E Q E 7 S, L, F, E S L 7 T, S T S 8 Q, Q, Y, D Q Q 8G, P P G 9 K, F, N, Q K Q 9 L, P L P 10 S, D, F, L F S 10 S S S 11 M, N,L, G M G 11 L L L 12 S, P, L S P 12 V, P P V 13 I, T, E, L, (V) I E 13K, V V K 14 (T, G) T L# 14 S, P S P 15 V, K, L L V 15 S, L L S 16 K, P,G G K 16 Q, G G Q 17 E, P E P 17 D, T D T 18 R, (H, G) R G 18 Q, L Q L19 V, A, (L) V A 19 A, S A S 20 S, S, H, (R, L) S S 20 L, S S L 21 I IV# 21 T, I I T 22 K, T, R T K 22 S, (R) S C# 23 I C* I 23 (S) C# S 24 S,K K S 24 R, V R V 25 P P C* 25 T S# T 26 K, (A), (S) S K 26 G, (R) R G27 Q, (A) Q A 27 D, Q Q D 28 N N S# 28 (S) S S 29 V, (G) V G 29 I, (L) LI 30 (Y), (T), (G) G Y 30 (T, V) V T *Cysteine sequence cannot bedetermined by N-terminal sequencing #Residues of discrepancy betweenn-terminal seq. results and hybridoma seq. results.

B. Cloning of Variable Regions of mAb into M13 Engineering Vector.

The DNA coding for the VL, C kappa, and VH regions of the mAbs (i.e.,8A10 and 3C6) were amplified with sequence specific primers using PCR.The resulting PCR product was gel-purified and restriction digested forspecific sites within the M13-based phage vector under the control oflacZ promoter. The double-stranded DNA coding for the VL, C kappa, andVH regions of 8A10 and 3C6 mAbs were ligated into the phage-based vectorcontaining the gene for the constant region of the CH1 region of a humanIgG1. The DNA was transformed into E. coli and the resulting plaquescontaining phage was examined in detail. Plaques were picked at random,their DNA isolated and the sequence determined. Clone 8A10_1 wasdetermined to have the identical sequence for the VL, C kappa, and VHregions as that of mAb 8A10 (not shown). The cloning process for 3C6 canbe performed according to the same protocol.

C. Demonstration of Paclitaxel Binding Activities of the Fabs Generatedfrom the M13 Engineering Vector.

A Clone 8A10_1 phage-infected culture of XL1-Blue E. coli(gram-negative) was grown while being induced with IPTG, was harvestedvia centrifugation and the periplasmic contents were released by osmoticshock (periprep). The released Fab products were isolated from theculture. The Fab concentration was quantified by ELISA and antigenspecific binding of the Fab was done using paclitaxel ELISA togetherwith intact IgGs (not shown). The K_(d) values (approximately 10 nM forboth antibodies) of the two intact IgGs were comparable to the onespreviously reported (see FIG. 3). The K_(d) value of Fab 8A10 wasdetermined to be 10 nM as well, which is the same as that of intact IgG8A10. These results suggest that the correct variable region sequence ofthe parental antibody 8A10 exists in the present Fab 8A10 and iscorrectly folded and expressed in the Fab format from the M13engineering vector.

The Fab expressed sufficiently well in the periplasmic prep todemonstrate concentration-dependent binding to BSA-paclitaxel conjugatecoated wells.

EXAMPLE 3 Representative Solid Phase Competitive Assay

In this example, a representative assay demonstrating the efficacy of asolid-phase competitive assay is described. The assay demonstrates theutility of using the anti-paclitaxel antibodies described herein in sucha detection format to provide informative signals for the present ofpaclitaxel in a sample. The results demonstrate that variable placementof the antibodies can enhance assay performance.

Paclitaxel lateral flow system. 1.2 mg/mL BSA-Pac (test lines, T) and0.2 mg/ml of goat-anti-mouse antibody (control line, C) were stripedonto a membrane card (high-flow plus HF180 membrane card, Millipore).Anti-paclitaxel antibody-colloidal gold conjugate was absorbed into andthe dried onto a conjugate pad (glass fiber pad, Millipore). Fetalbovine serum (FBS) spiked with paclitaxel (10 uL), chased by 80 μL ofPBS Tween, was flowed in the assay.

Tandem Antibody Assay. The antibody-gold conjugates are reconstitutedusing distilled water and are then added to each other to make theappropriate concentrations. This tandem antibody solution is applied andthen dried onto the assay conjugate pads.

Reader Output: Intensity vs Position. Readout of the results of scanningthe test strips. The strips were read using Qiagen reader (Qiagen,Germany).

Paclitaxel Standard Curve. Standard curves of ratio of test line overcontrol line vs. paclitaxel concentration were generated.

FIGS. 5A and 5B illustrate curves for 8A10 bound at lines T1 and T2.FIG. 5A illustrates the standard curve, i.e., the ratio of test lineover control line (T/C) vs. paclitaxel concentration. The largedifference in ratio for 8A10 at T1 versus T2 for the lowerconcentrations indicates a much higher sensitivity for the antibody whenplaced closer to the sample port, where concentration of analyte islikely to be higher. FIG. 5B illustrates the output intensity vs.position readout of scanned test strips as provided by a reader device.

FIGS. 6A and 6B illustrate curves for 3C6 bound at lines T1 and T2. FIG.6A illustrates the standard curve, i.e., the ratio of test line overcontrol line (T/C) vs. paclitaxel concentration. The relatively minordifference in ratio for 3C6 at T1 versus T2 for the lower concentrationsindicates a relatively low improvement in sensitivity would be gainedfor placing the antibody closer to the sample port, where concentrationof analyte is likely to be higher. However, improvement in signalintensity relative to at T2 was observed. FIG. 6B illustrates the outputintensity vs. position readout of scanned test strips as provided by areader device.

FIGS. 7A and 7B illustrate curves for combined 8A10 and 3C6 bound atlines T1 and T2. FIG. 7A illustrates the standard curve, i.e., the ratioof test line over control line (T/C) vs. paclitaxel concentration. Theassay was made more robust by combining the high sensitivity of 8A10close to the sample port and the higher sensitivity of 3C6 at T2 that islocation independent. FIG. 7B illustrates the output intensity vs.position readout of scanned test strips as provided by reader device.

In the above analyses (and in FIGS. 5-7), the measurement of position ofT1, T2, and C (Pos [mm]) in FIGS. 5B, 6B, and 7B was made from thedownstream end in flow direction (e.g., sample introduced at 55 mmpoint, T2 at about 45 mm, T1 at about 40 mm, and C at about 35 mm) ofthe test strip.

EXAMPLE 4 Method for Determining Antibody Binding Properties

In this example, a method for determining antibody properties isdescribed. The methods were carried out by Precision Antibody, Inc.(Columbia, Md.) using a Biacore Assay. This example also shows how theLFA assay of the invention can be adapted to other solid phase assays,such as Surface Plasmon Resonance (SPR) assays.

Binding experiments were performed on a Biacore 3000 instrument (GE,Pittsburgh, Pa.) at 25° C. Approximately 13,000 RU of anti-BSA ab (LifeTechnologies, A11133, lot 1637270) was directly immobilized on flow cell2 of CM5 chip by amine coupling (EDC/NHS). 110-120 RU of BSA-paclitaxel(BSA-Ag) was captured. Flow cell 1 was treated the same way but withoutthe ligand and used as a blank surface for reference subtraction. Theunoccupied sites were blocked with 1 M ethanol amine. Analytes Ab 1(8A10) and Ab2 (3C6) were flowed over the chip at variableconcentrations. Binding of antigen to the antibodies was monitored inreal time to obtain on (k_(a)) and off (k_(d)) rates. The equilibriumconstant (K_(D)) was calculated from the observed k_(a) and k_(d).

Full kinetic analysis was performed using analyte concentrations asindicated with 2-fold serial dilutions. The starting concentration was200 nM, followed by 100, 50, 25, 12.5, and 0 nM. The 100 nMconcentration was run in duplicates to confirm the reproducibility ofthe assay. Full kinetic analysis results are summarized in Table 3.

The assay buffer was 10 mM HEPES buffer (pH 7.4), 150 mM NaCl, 3 mMEDTA, 0.05% P20 (polyoxyethylenesorbitan). The regeneration buffer was10 mM Glycine buffer (pH 2.0). The conjugation buffer was 10 mM sodiumacetate buffer (pH 5.0). The flow rate used for capturing the ligand was1 μL/min. The flow rate for kinetics analysis was 50 μL/min.

Chi square (χ²) analysis was carried out between the actual sensorgramand the sensorgram generated from the BlAnalysis software to determinethe accuracy of the analysis. A χ² value within 1-2 is consideredsignificant (accurate) and below 1 is highly significant (highlyaccurate).

A summary of three independent SPR runs are presented in Table 4.

TABLE 3 Full Kinetic Analysis. k_(a) K_(d) K_(A) K_(D) Con LigandAnalyte (1/Ms) (1/s) R_(max) (1/M) (M) (nM) χ² BSA-Ag Ab1 4.70 × 10⁵3.04 × 10⁻⁴ 4.01 1.55 × 10⁹ 6.45 × 10⁻¹⁰ 0-100 0.173 (110RU) BSA-Ag Ab22.05 × 10⁴ 9.67 × 10⁻⁴ 15.6 2.12 × 10⁷ 4.72 × 10⁻⁸ 0-200 0.179 (110RU)

TABLE 4 SPR Summary. Antibody Instrument On the Chip On-rate Off-rateK_(d) R_(max) Note 8A10 Biacore 3000 Antibody 1.6 × 10⁶ 2.0 × 10⁻³ 1.3 ×10⁻⁹ 3.6 Biacore T-200 Antibody 2.4 × 10⁵ 8.7 × 10⁻⁴ 3.6 × 10⁻⁹ 8.6Biacore 3000 Antigen 4.7 × 10⁵ 3.0 × 10⁻⁴ 6.5 × 10⁻¹⁰ 4.0 3C6 Biacore3000 Antibody 2.8 × 10⁴ 2.9 × 10⁻⁴ 1.0 × 10⁻⁸ 54.4 Single point BiacoreT-200 Antibody 1.8 × 10⁴ 2.2 × 10⁻⁴ 1.2 × 10⁻⁸ 112 Biacore 3000 Antigen2.1 × 10⁴ 9.7 × 10⁻⁴ 4.7 × 10⁻⁸ 15.6

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method for assayingpaclitaxel in a liquid sample, comprising: (a) applying a liquid samplecomprising paclitaxel to a lateral flow assay device, the devicecomprising (i) a sample receiving zone for receiving the liquid sample;(ii) a detection reagent zone in liquid communication with the samplereceiving zone and downstream in flow direction from the samplereceiving zone, wherein the detection reagent zone comprises a detectionreagent deposited thereon, wherein the detection reagent is a paclitaxelantibody, or fragment or derivative thereof, that binds paclitaxel,labeled with a detectable reporting group, and wherein the paclitaxelantibody, or fragment or derivative thereof, has a K_(on) from about 10⁴to about 10⁷, and a K_(off) from about 10⁻³ to about 10⁻⁷; and (iii) acapture zone in liquid communication with the detection reagent zone anddownstream in flow direction from the detection reagent zone, whereinthe capture zone comprises first and second capture reagents immobilizedthereon, wherein the first capture reagent is a paclitaxel materialcapable of binding the detection reagent (test line), wherein the secondcapture reagent is an antibody capable of binding the detection reagent(control line), wherein the first capture reagent is positioned at afirst distance downstream in flow direction from the upstream end of thecapture zone, wherein the second capture reagent is positioned at asecond distance downstream in flow direction from the upstream end ofthe capture zone, wherein the second distance is greater than the firstdistance, and wherein the ratio of the first distance to the seconddistance is from about 0.0 to about 0.4, when the K_(on) is greater thanabout 2.0×10⁵ and the K_(off) is less than about 1.0×10⁻³, and whereinthe ratio of the first distance to the second distance is from about 0.2to about 1.0, when the K_(on) is greater than about 2.0×10⁴ and theK_(off) is less than about 2.0×10⁻⁴; and (b) allowing the sample to flowfrom the sample receiving zone through the detection reagent zone toprovide detection reagent with paclitaxel; (c) allowing the detectionreagent with paclitaxel to flow through the capture zone, whereby thefirst capture reagent (test line) competes with the analyte (paclitaxel)for binding with the detection reagent, and whereby the second capturereagent (control line) binds excess detection reagent; and (d) observingthe amount of detection reagent bound to the first capture reagent (testline) relative to the second capture reagent (control line).
 2. Themethod of claim 1 further comprising determining the quantity ofpaclitaxel in the sample by quantitating the amount of detection reagentbound at control line and test line.
 3. The method of claim 2, whereinquantitating the amount of detection reagent bound to the capturereagents comprises optical density measurement.
 4. The method of claim1, wherein the detectable reporting group is colloidal gold.
 5. Themethod of claim 1, wherein the paclitaxel antibody is 3C6.
 6. The methodof claim 1, wherein the paclitaxel antibody is 8A10.
 7. The method ofclaim 1, wherein the paclitaxel material is a paclitaxel antigen thatcompetes with paclitaxel for binding to the detection reagent.
 8. Themethod of claim 1, wherein the paclitaxel material is a paclitaxelprotein conjugate.
 9. The method of claim 1, wherein the distancebetween the sample receiving zone and the first capture reagent isvaried to optimize paclitaxel detection sensitivity.
 10. The method ofclaim 1, wherein the distance between the sample receiving zone and thefirst capture reagent is minimized to optimize paclitaxel detectionsensitivity.
 11. The method of claim 1 further comprising observing theamount of excess detection reagent bound to the second capture reagent(control line).
 12. The method of claim 1 further comprising determiningthe quantity of paclitaxel in the sample by quantitating the amount ofexcess detection reagent bound to the second capture reagent.
 13. Themethod of claim 1 further comprising a third capture zone intermediatethe first and second capture zones, wherein the third capture zonecomprises a paclitaxel material capable of binding the detectionreagent.
 14. The method of claim 13, comprising determining the quantityof paclitaxel in the sample by quantitating the amount of detectionreagent bound to the third capture reagent.
 15. The method of claim 13,wherein quantitating the amount of detection reagent bound to the thirdcapture reagent comprises optical density measurement.
 16. The method ofclaim 1, wherein first and second antibodies are used, and wherein thefirst has a K_(on) greater than 10⁴ and the second has a K_(off) lessthan 10⁻³.
 17. The method of claim 1, wherein two or three lines can beused to generate multiple readings on the same sample, allowing forincreased reproducibility and expanded dynamic range.
 18. A method formonitoring the efficacy of a paclitaxel therapy in a patient diagnosedwith cancer, comprising: (a) treating a cancer patient with paclitaxelat a first point in time; (b) determining a first concentration ofpaclitaxel in the patient at a first point in time, wherein determiningthe concentration comprises the method of claim 1; (c) treating thepatient with paclitaxel at a second point in time; (d) determining asecond concentration of paclitaxel drug in the patient at a second pointin time, wherein determining the concentration comprises the methods ofclaim 1; and (e) comparing the first and second concentrations ofpaclitaxel in the patient to determine the efficacy of the cancertreatment.
 19. A method for PK-guided dosing of paclitaxel therapy in apatient diagnosed with cancer, comprising: (a) treating the cancerpatient with paclitaxel at a first point in time; (b) determining one ormore pharmacokinetic parameters of paclitaxel in the patient at a firstpoint in time, wherein determining the one or more pharmacokineticparameters comprises measuring the concentration of paclitaxel by themethod of claim 1; (c) treating the patient with paclitaxel at a secondpoint in time using the PK information from the first dosing; (d)determining one or more pharmacokinetic parameters of paclitaxel in thepatient at a second point in time, wherein determining the one or morepharmacokinetic parameters comprises measuring the concentration ofpaclitaxel by the method of claim 1; and (e) comparing one or morepharmacokinetic parameters of paclitaxel in the subject at the firstpoint in time with the levels at the second point in time to confirmthat optimal dosing was achieved.
 20. The method of claim 19, whereinthe pharmacokinetic parameters are selected from the group consisting oftime to maximum concentration (T_(max)), concentration maximum(C_(max)), area under the curve (AUC), clearance (CL), volume ofdistribution (V_(d)), apparent volume of distribution during theterminal phase (V_(Z)), apparent volume of distribution during steadystate (V_(ss)), and combinations thereof.